Broad-spectrum therapeutics against influenza virus infections are critically needed to address the problem of influenza pandemics, a major threat to the public health globally. Interfering with virus entry is a novel and attractive therapeutic strategy to control virus infection. Proof of principle of this approach has come from the HIV inhibitor enfuvirtide (T-20). Our goal is to discover nonpeptidic small molecules that will inhibit entry of avian influenza H5N1 and other potentially pandemic influenza viruses. Our strategy is to target envelope glycoprotein hemagglutinin (HA), which mediates influenza virus entry through receptor binding and fusion with host cells. HA is class I fusion protein like the HIV Gp120 and F protein of paramyxoviruses. The class I virus fusion proteins undergo a series of conformational rearrangements during fusion that leads to fusion hairpin structure. This resultant structure promotes the juxtaposition of the viral and cellular envelopes during fusion and is sustained by protein protein interactions. Small molecule entry inhibitors of paramyxoviruses have been identified that interfere with the formation of this fusion hairpin structure. Because analogous structures are present in HA and other class I fusion proteins, the results support our hypothesis of targeting HA as a strategy for drug discovery. Inhibitors targeting this conserved site will be active against multiple subtypes, including a newly emerged pandemic strain. We will use a pseudotype virus expressing HA (H5 subtype), which has been developed as a surrogate model, to mimic HA mediated entry and screen for entry inhibitors under BSL2 conditions. In preliminary studies, we have developed and characterized a sensitive pseudotype virus assay for screening HA inhibitors. In Phase I of this STTR project, we will optimize the assay for rapid screening of a large (>100,000) library of structurally diverse small molecules. Hits will be confirmed in a blind fashion by our collaborator Dr Lijun Rong, University of Illinois at Chicago and will be evaluated for their anti-influenza activity against live H5 avian influenza viruses in an enhanced BSL 3 laboratory by Dr Adolfo Garcia-Sastre, at Mount Sinai Medical College. Confirmed hits will be evaluated for their spectrum against other subtypes and prioritized based on their mechanism of action. These novel influenza therapeutics will block virus entry and suppress cellular cytotoxicity resulting from virus-cell contact. They will also be expected to have a low incidence of resistance development since minor deviations in the conserved domains would prevent fusion of virus with endosomal membrane. In Phase II, we will progress the most promising scaffolds through a rational drug design program, and will test lead compounds for efficacy and toxicity in animal models. The most active compound, with the least toxicity, will advance to IND enabling studies. PUBLIC HEALTH RELEVANCE: Influenza is a highly infectious acute respiratory disease, characterized by recurrent annual epidemics and periodic major worldwide pandemics. Vaccines, currently the primary strategy for protection against influenza virus infection, are only effective if they match the circulating virus type(s) and cannot be developed in advance against new emerging pandemic strain(s). Our goal is to develop an anti-influenza therapeutic that will prevent virus entry by targeting the conserved fusion and receptor binding domains of envelope protein hemagglutinin (HA), and will be active against all subtypes, including a newly emerging pandemic strain.